A Thermophilic Gram-Negative Nitrate-Reducing Bacterium

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A Thermophilic Gram-Negative Nitrate-Reducing Bacterium, Calditerrivibrio nitroreducens, Exhibiting Electricity Generation Capability Qian Fu,† Hajime Kobayashi,*,†,‡ Hideo Kawaguchi,† Tatsuki Wakayama,§ Haruo Maeda,§ and Kozo Sato*,†,‡ †

Department of Systems Innovation, Graduate School of Engineering, The University of Tokyo, Tokyo, Japan Engineering for Sustainable Carbon Cycle (INPEX Corporation) Social Cooperation Program, Frontier Research Center for Energy and Resource (FRCER), The University of Tokyo, Tokyo, Japan § INPEX Corporation, 9-23-30 Kitakarasuyama, Setagaya-ku, Tokyo 157-0061, Japan ‡

S Supporting Information *

ABSTRACT: To exploit the potential diversity of thermophilic exoelectrogens, two-chamber microbial fuel cells (MFCs) were inoculated with thermophilic anaerobic digester sludge and operated at 55 °C without supplementing with exogenous redox mediator. The MFC generated a maximum power density of 823 mW m−2 after 200 h of operation. Molecular phylogenetic analyses suggested that the microbial population on the anode was dominated by a species closely related to a thermophilic nitrate-reducing bacterium Calditerrivibrio nitroreducens, for which a strain (Yu37-1) has been isolated in pure culture. Thus, a pure culture of the C. nitroreducens strain Yu37-1 was inoculated into MFC to examine the electricity generation capability. Without an exogenous mediator, MFCs stably produced electricity with a maximum power density of 272 mW m−2 for >400 h of operation. The MFC current recovered to the original level within few hours after medium replacement, suggesting that the electricity generation was caused by the anodic microorganisms. Cyclic voltammetry indicated that redox systems (E3 and Ec) with similar potentials (−0.14 and −0.17 V) made the main contributions to the exoelectrogenic activities of the sludge-derived consortium and C. nitroreducens Yu37-1, respectively. This study undertook the bioelectrochemical characterization of C. nitroreducens as the first example of a thermophilic Gram-negative exoelectrogen.

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using membrane-bound c-type cytochromes8,10 and conductive pili.6,7 Membrane-bound c-type cytochromes14,15 and conductive pili12,16 have similarly been characterized in S. oneidensis. However, the extracellular electron transfer of S. oneidensis is also mediated by redox-active flavin compounds,11,13 which are secreted by the bacteria and function as electron shuttles. In contrast to the mesophilic species, thermophilic members of exoelectrogens remain underexploited and have so far been largely limited to the Gram-positive species affiliated with the genus Thermincola of the phylum Firmicutes. Two thermophilic bacteria strains, T. potens strain JR17 and T. ferriacetica strain Z0001,18 have been shown to transfer electrons, in the absence of exogenous mediators, directly to an electrode. A recent study has suggested that multiheme c-type cytochromes localized to

INTRODUCTION The microbial fuel cell (MFC) is a promising energy generation/conversion technology that can be used in renewable energy generation, wastewater treatment, biosensing, and bioremediation.1−3 A key feature of MFCs is the use of microorganisms (collectively called “exoelectrogens”) possessing the capability of extracellularly transferring electrons to electrodes, as biocatalysts to harvest energy from organic materials, including various types of wastes and biomass. In a typical MFC, exoelectrogens anaerobically oxidize organic matter and extracellularly release electrons (as the terminal step of anaerobic respiration), which are collected at an anode for current generation.1−3 To date, exoelectrogenic activity has been reported for >20 microbial species, most of which are mesophilic Gram-negative bacteria affiliated with the phylum Proteobacteria.1,4 Among them, Geobacter sulfurreducens5−10 and Shewanella oneidensis11−16 have been extensively studied as model exoelectrogens. Biochemical and genetic studies have indicated that G. sulfurreducens can directly transfer electrons to an anode © XXXX American Chemical Society

Received: June 21, 2013 Revised: September 16, 2013 Accepted: September 20, 2013

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the cell envelope of the T. potens strain JR, which in contrast to the Gram-negative envelope lacks an outer membrane, but instead has a cell wall, are implicated in the electron transfer.19 However, molecular phylogenetic analyses have suggested that more diverse species of thermophilic bacteria share exoelectrogenic activity. In thermophilic MFCs inoculated with marine sediment- and digester sludge-derived consortia, although Firmicutes (particularly, genus Thermincola)-affiliated species dominated the anodic microbial communities, sequences related to other phyla (including Deferribacteres, Proteobacteria, Spirochaetae, Nitrospirae, Thermotogae, and Coprothermobacter) were also detected.17,20 Moreover, in a thermophilic MFC continuously fed with distillery wastewater, a Bacteriodetes bacterium was the dominant species in the anode and Firmicutes was the second most populated group.21 Furthermore, in a digester effluent-inoculated thermophilic MFC, no Firmicute-related sequence was detected at the anode and the dominant species belonged to the phyla Deferribacteres and Coprothermobacter.22 However, the exoelectrogenic activity of such non-Firmicute thermophiles has never been investigated in pure culture. It has recently been suggested that thermophilic MFCs are potentially superior to mesophilic MFCs in performance, with higher reaction activity, greater durability, and a wider substrate range.17,20,23 With regard to possible applications under diverse conditions, the availability of more biocatalysts (thermophilic exoelectrogens in addition to Thermincola-affiliated species) will improve the performance of thermophilic MFCs. We accordingly sought to investigate new thermophilic exoelectrogens. A two-chamber MFC was inoculated with thermophilic anaerobic digester sludge without exogenous mediators and showed substantial current generation at 55 °C. Phylogenetic analysis suggested that the anode biofilm was dominated by a bacterium closely related to Calditerrivibrio nitroreducens, a thermophilic nitrate-reducing bacterium, for which a strain (Yu37-1) is available in pure culture. Finally, we demonstrated the electricity generation ability of the strain, expanding members of thermophilic exoelectrogens to Gram-negative species.

The voltage (E) across the external resistance was automatically monitored every 5 min using a data acquisition unit (Agilent 34970A; Agilent Technologies). Each reactor was operated in a fed-batch mode continuously stirred with a magnetic stir bar and incubated at 55 °C except for the room-temperature control. Electrochemical Analyses. The polarization and power density curves as a function of current were obtained using a variable resistance box (10000−30 Ω). The voltage (E) across each external resistance was measured with the Agilent 34970A. The current (I) was calculated according to Ohm’s law: I = E/ R, and the power density (PD) was calculated according to PD = EI/A, where R (Ω) represents the external resistance and A (m2) represents the surface area of the anode. All the potentials reported in this study are given in volts (V) relative to the standard hydrogen electrode (SHE). Cyclic voltammetry (CV) was performed using a potentiostat (HSV-110, Hokuto Denko, Japan) with a standard threeelectrode system. The anode and the cathode (ferricyanide as the catholyte) acted as the working electrode and the counter electrode, respectively. As the reference electrode, an Ag/AgCl reference electrode was inserted into the anode chamber. In turnover conditions, the parameters for CV were: equilibrium time 99 s (at −0.4 V), scan rate 1 mV s−1, and scan range from −0.4 to 0.2 V. The anaerobic medium lacking soluble redox compounds was used in the anode chamber. CV measurements with cell-free spent medium and noninoculated control medium were also performed using a presterilized electrode in the same reactor. The spent medium was collected from the anode chamber and then filtered with a presterilized filter in an anoxic chamber to remove planktonic cells. The filtrates were then analyzed to determine whether soluble electron shuttles were present in the spent medium. In nonturnover conditions, the anode was washed thrice with acetate-free fresh medium and then poised at −0.1 V until the current fell approximately to the background level (2 μA cm−2), after which CV was performed with increasing scan rates from 1 mV s−1 to 1 V s−1 (scan range: −0.5 to 0.3 V). Characterization of the Anodic Bacterial Population. For scanning electron microscopy, anodes were aseptically sliced and fixed by 2.5% (w/v) glutaraldehyde and 2% (w/v) paraformaldehyde in 0.1 M phosphate buffer solution (pH 7.4). For microbial population analysis, community DNA was directly extracted from 250 mg of aseptically crushed anode using a PowerSoil DNA isolation kit (MO BIO laboratories). The extracted DNA (20 ng) was used as the template for PCR with the primers 8F (5′-AGAGTTTGATYMTGGCTCAG-3′) and 1492R (5′-CGGYTACCTTGTTACGACTT-3′).26 To minimize PCR bias, the number of PCR cycles was minimized to 16 cycles and PCR products of three independent reactions were pooled.27−29 The pooled PCR amplicons were cloned into pCR4−TOPO using a TOPO TA cloning system (Invitrogen, Carlsbad, CA) according to the manufacturer’s instructions. Plasmids were purified using a high pure plasmid isolation kit (Roche Applied Science, Indianapolis, IN) and sequenced with T3 and T7 primers. Sequences of 95 clones were analyzed until Good’s coverage estimator30 reached 97%. The assembled sequences were aligned with the NAST aligner program in Greengenes (http://greengenes.lbl.gov/) with the closest sequence relatives from the NCBI database in October 2012. The alignments were then manually improved using MEGA 4.0.2.31 A total of 1127 nucleotide positions were used in the alignments. A phylogenetic tree was constructed on the basis of

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MATERIALS AND METHODS MFC Construction. Two-chamber MFCs were constructed, each comprising two glass bottles (volume, 300 mL) separated by a proton exchange membrane (Nafion 117, DuPont Co.). The proton exchange membranes were pretreated as previously described.24 Both anode and cathode were made of plain carbon cloth (2 × 10 cm, TMIL Ltd.). Titanium wires (0.5 mm, Alfa Aesar) were used to connect the electrodes to circuits. The internal resistances between the electrodes and titanium wires were 20 mV s−1 (SI Figure S3), suggesting that the electron-transfer regime governing the pure-culture system was rather complex than the classical thin-film behavior reported for purified enzymes or chemical catalysts.41 In contrast, at the sludge-inoculated MFC anode, the catalytic voltammetric behavior showed a strong dependence on the scan rate. Figure 7 depicts the dependency of the E3

Figure 6. Cyclic voltammetry analyses of the MFC anode inoculated with the C. nitroreducens strain Yu37-1: (A) A turnover CV using a scan rate of 1 mV s−1 and its first derivative (inset); (B) A nonturnover CV, scan rate: 1 mV s−1; (C) Plot of peak current vs scan rate of the nonturnover CV. CV was measured in triplicate and each panel shows the data from one representative experiment.

Figure 7. Scan rate analyses of the sludge-inoculated MFC anode in nonturnover conditions: (A) Plot of peak current vs scan rate; (B) Plot of peak current vs square root of scan rate.

peak current on the scan rate (v) (Figure 7A) and its square root (v1/2) (Figure 7B). The plots showed that the peak current was linear up to a threshold scan rate of 10 mV s−1 (Figure 7A). However, at a scan rate >10 mV s−1, the peak current was proportional to v1/2 (Figure 7B), indicating a diffuioncontrolled regime.43,44 Consistent with this result, drastic peak potential shift was observed at a scan rate >10 mV s−1 (SI Figure S4). Such bimodal behavior had also been reported in the MFC anode inoculated with G. sulfurreducens, which formed electroactive biofilm on the electrode surface.8 Based on electrochemical analyses of the G. sulf urreducens−inoculated anode, a model has been proposed for current generation at a biofilm-modified anode.8,45 Considering a multilayered biofilm developed on the surface (Figure 4A), it was reasonable to speculate the model could be applied to the sludge-inoculated MFC anode.45 The model suggested that the bimodal behavior was due to confined diffusion of redox mediators and/or charge-compensating ions in the biofilm:8 Slow scan rates allowed enough time for diffusion through the biofilm of either soluble mediators or charge-compensating ions to occur. Thus, at scan rates slower than 10 mV s−1, current was not limited by diffusion and showed a linear dependency to the scan rate. The

CV. In the anode of the C. nitroreducens-inoculated MFC, the peak current of system Ec was directly proportional to the scan rate (v) for all values of the scan rate examined (1−100 mV s−1) (Figure 6C). Such linear dependence of peak height on scan rate was typically considered as indicative of “thin-film behavior”, where the “film” released (or accepted) electrons as a single entity. Thin-film behavior has commonly been reported for redox proteins adsorbed on electrode surfaces.41 With regard to microbial electrochemical system, submonolayer films of S. oneidensis cells adsorbed on electrode surfaces have shown thin-film behavior in the absence of both electron donors and electron shuttles.42 Based on the sparse colonization of C. nitroreducens strain Yu37-1 on the anode surface (Figure 4B), we speculated that the C. nitroreducens cells behaved like a thin (submonolayer) film in electrode reactions, as was evident in the scan rate analysis (Figure 6C). Thus, it was suggested that, in the C. nitroreducens-inoculated MFC anode, the interfacial electron transfer (the final hop of electrons from the redox proteins to the electrode) was slower than intracellular reaction(s) feeding electrons to the interface (redox proteins on the cell surface), being the rate-limiting step for current F

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Young Scientists (B) 23780074 (to H.K.). The authors thank Dr. Hiroshi Sagara (The Institute of Medical Science, the Univirsity of Tokyo) for SEM analysis.

sludge-derived biofilm contained little extracellular polymeric substances and consisted mostly of “bare” microbial cells (Figure 4A). We speculated that such biofilm might allow solute diffusion through biofilm to a relatively greater extent than biofilms containing dense extracellular matrix (such as the Geobacter biofilm46) did. At a scan rate >10 mV s−1, however, as there was less time for the diffusion to occur, the diffusion of soluble mediator or charge-compensating ions limited the current generation, resulting in a linear dependency of the peak current to v1/2.8 As no soluble mediator was detected in the spent medium (Figure 5A), it was likely that chargecompensating ions (such as protons) were responsible for the diffusional limitaion to electron transfer, as also suggested in the G. sulfurreducens−inoculated anode. Those hypotheses will be examined by more detailed electrochemical analyses of the anodes in future study. Thus, the present study investiagted a new thermophilic exoelectrogen, C. nitroreducens strain Yu37-1, a Gram-negative nitrate-reducing bacterium of the Deferribacteres phylum. CV analyses and medium replacement experiments showed that the pure culture was capable of producing electricity in a direct electron transfer manner. However, the molecular species corresponding to the redox system (Ec) remain to be determined. The genome of the C. nitroreducens strain Yu37-1 encodes 12 hypothetical proteins containing at least one c-type cytochrome domain.47 Among them, the protein encoded by ORF Calni_1470 is a candidate involved in the redox system, as it contains a multiheme c-type cytochrome domain and shares limited but significant primary sequence similarity (with E-value of 2 × 10−12) with TherJR_1117, the multiheme c-type cytochrome involved in the extracellular electron transfer in the T. potens strain JR.19 Homologues of the ORF Calni_1470 are interestingly also found in genomes of other bacteria of the Deferribacteres phylum (such as Deferribacter desulf uricans SSM1, Flexistipes sinusarabici MAS10, and Denitrovibrio acetiphilus DSM 12809). In particular, given that two species of the Deferribacter genus (a genus closely related to Calditerrivibrio), D. thermophilus48 and D. abyssi,49 have been reported to be capable of reducing insoluble iron, it is possible that more diverse bacteria affiliated to the Deferribacteres phylum also have exoelectrogenic activity.

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Additional information as noted in the text. This material is available free of charge via the Internet at http://pubs.acs.org.

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Corresponding Authors

*(H.K.) Phone: +81-(3)-5841-7041; fax: +81-(3)-3818-7492; e-mail: [email protected]. *(K.S.) E-mail: [email protected] Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The Engineering for Sustainable Carbon Cycle (INPEX Co.) Social Cooperation Program is a social cooperation program of the University of Tokyo with INPEX Co., and financed by INPEX Co. This work was supported by Japan Society for the Promotion of Science (JSPS) Grant-in-Aid for Scientific Research (A) 20246128 (to K.S.) and a Grant-in-Aid for G

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